In the course of studies of platelet-derived growth factor (PDGF) isoforms, a novel, cysteine-rich mitogenic peptide secreted by human vascular endothelial cells and related to the v-src-induced immediate early gene product CEF-10 was identified. An anti-PDGF antibody was used to screen a human umbilical vein endothelial cell (HUVEC) expression library, and the gene encoding this novel mitogen was named connective tissue growth factor (CTGF). The connective tissue growth factor protein was shown to stimulate DNA synthesis and promote chemotaxis of fibroblasts (Bradham et al., J. Cell Biol., 1991, 114, 1285-1294).
Connective tissue growth factor (CTGF; also known as ctgrofact, fibroblast inducible secreted protein, fisp-12, NOV2, insulin-like growth factor-binding protein-related protein 2, IGFBP-rP2, IGFBP-8, HBGF-0.8, Hcs24, and ecogenin) is a member of the CCN(CTGF/CYR61/NOV) family of modular proteins, named for the first family members identified, connective tissue growth factor, cysteine-rich (CYR61), and nephroblastoma overexpressed (NOV), but the family also includes the proteins ELM-1 (expressed in low-metastatic cells), WISP-3 (Wnt-1-induced secreted protein), and COP-1 (WISP-2). CCN proteins have been found to be secreted, extracellular matrix-associated proteins that regulate cellular processes such as adhesion, migration, mitogenesis, differentiation, survival, angiogenesis, atherosclerosis, chondrogenesis, wound healing, tumorigenesis, and vascular and fibrotic diseases like scleroderma (Lau and Lam, Exp. Cell Res., 1999, 248, 44-57).
In most cases, a single 2.4-kilobase connective tissue growth factor transcript has been reported in expression studies, although 3.5- and 7-kilobase transcripts have been reported in glioblastoma cells. Connective tissue growth factor is expressed in fibroblasts during normal differentiation processes that involve extracellular matrix (ECM) production and remodeling, such as embryogenesis and uterine decidualization following implantation. Connective tissue growth factor is also frequently overexpressed in fibrotic skin disorders such as systemic sclerosis, localized skin sclerosis, keloids, scar tissue, eosinophilic fasciitis, nodular fasciitis, and Dupuytren's contracture. Connective tissue growth factor mRNA or protein levels are elevated in fibrotic lesions of major organs and tissues including the liver, kidney, lung, cardiovascular system, pancreas, bowel, eye, and gingiva. In mammary, pancreatic and fibrohistiocytic tumors characterized by significant connective tissue involvement, connective tissue growth factor is overexpressed in the stromal compartment. In many cases, connective tissue growth factor expression is linked spatially and temporally to the profibrogenic cytokine transforming growth factor-beta (TGF-β) (Moussad and Brigstock, Mol. Genet. Metab., 2000, 71, 276-292).
Connective tissue growth factor has been mapped to human chromosomal region 6q23.1, proximal to the c-myb gene, and chromosomal abnormalities involving this region have been associated with human tumors, such as Wilms' tumor (Martinerie et al., Oncogene, 1992, 7, 2529-2534).
Tumors with significant fibrotic and vascular components exhibit increased connective tissue growth factor expression, and connective tissue growth factor may be involved in the pathogenesis of pediatric myofibroblastic tumors. Of 12 pediatric tumors examined, all showed moderate to intense connective tissue growth factor expression in tumor cells and/or endothelial cells of the associated vasculature (Kasaragod et al., Pediatr. Dev. Pathol., 2001, 4, 37-45).
Connective tissue growth factor mRNA is also specifically upregulated in malignant human leukemic lymphoblasts from children with acute lymphoblastic leukemia (ALL) (Vorwerk et al., Br. J. Cancer, 2000, 83, 756-760), and both mRNA and protein levels are upregulated by TGF-beta in Hs578T human breast cancer cells in a dose-dependent manner, indicating that connective tissue growth factor is an important neuroendocrine factor and a critical downstream effector of TGF-beta (Yang et al., J. Clin. Endocrinol. Metab., 1998, 83, 2593-2596).
Based on a region of amino acid homology to insulin-like growth factor (IGF) binding proteins (IGFBPs), connective tissue growth factor was hypothesized to regulate cell growth through IGF. Recombinant human connective tissue growth factor was expressed in a baculoviral system and demonstrated to bind to IGF in vitro with low affinity, and thus, connective tissue growth factor was identified as a member of the IGFBP superfamily, and was given the name IGFBP-8 (Kim et al., Proc. Natl. Acad. Sci. U.S.A., 1997, 94, 12981-12986).
The role of connective tissue growth factor has been investigated in a transgenic mouse. Transgenic mice overproducing the connective tissue growth factor protein under control of the collagen promoter could develop and their embryonic and neonatal growth were normal, but they displayed dwarfism within a few months of birth, bone density was decreased compared with normal mice, male testes were much smaller than normal and fertility was affected. These results indicate that the effects of overexpression of connective tissue growth factor affects endochondral ossification, and may also regulate embryonic development of the testes (Nakanishi et al., Biochem. Biophys. Res. Commun., 2001, 281, 678-681).
In cultured 22-day fetal rat calvarial osteoblasts, cortisol stimulates transcription of connective tissue growth factor in a time- and dose-dependent manner, and cyclohexamide did not preclude this effect, indicating that this upregulation was not protein synthesis dependent. Glucocorticoids have complex effects on bone, some due to direct regulation of specific genes expressed by osteoblasts, and some indirect, mediated by locally produced growth factors or their binding proteins. IGFs have important stimulatory effects on bone formation, but glucocorticoids inhibit expression of IGFs. Because connective tissue growth factor binds to IGF, its increased expression could modulate the effect of cortisol on bone (Pereira et al., Am. J. Physiol. Endocrinol. Metab., 2000, 279, E570-576).
Connective tissue growth factor may be regulated not only at the level of transcription, but also by proteolytic degradation, but this varies with cell type. In large vessel bovine endothelial cells, cyclic AMP (cAMP) was found to increase expression of intact connective tissue growth factor protein by inhibiting degradation, whereas TGF-beta stimulated neither mRNA nor protein levels. In microvessel cells, TGF-beta stimulates an increase in connective tissue growth factor mRNA and both TGF-beta and cAMP stimulated proteolytic degradation, and these differences may have an effect on angiogenesis and wound healing (Boes et al., Endocrinology, 1999, 140, 1575-1580).
Purified murine connective tissue growth factor promotes the adhesion of primary human dermal microvascular endothelial cells (HMVECs) and of platelets through integrin receptors αvβ3 and αIIbβ3, respectively, suggesting its involvement in cell adhesion signaling, hemostasis and thrombosis (Babic et al., Mol. Cell. Biol., 1999, 19, 2958-2966; Jedsadayanmata et al., J. Biol. Chem., 1999, 274, 24321-24327). Connective tissue growth factor also stimulates migration of HMVECs in culture through an integrin receptor α3β3− dependent mechanism. Furthermore, connective tissue growth factor can promote survival of HMVECs plated onto laminin but deprived of growth factors, a condition that otherwise induces apoptosis. In vivo, connective tissue growth factor induces neovascularization in rat corneal micropocket implants. Thus, connective tissue growth factor is an angiogenic inducer, and may play a role in adhesion, migration, and survival of endothelial cells during blood vessel growth, perhaps by delivering an antiapoptotic signal via its interaction with integrin αvβ3 (Babic et al., Mol. Cell. Biol., 1999, 19, 2958-2966).
In contrast, connective tissue growth factor may negatively regulate growth of human prostate cells. Connective tissue growth factor expression is upregulated during senescence of normal human prostate epithelial cells (HPECs), and connective tissue growth factor is responsive to growth regulators such as all-trans retinoic acid (atRA), supporting a growth-regulatory role of connective tissue growth factor in prostatic epithelium (Lopez-Bermejo et al., Endocrinology, 2000, 141, 4072-4080).
Expansion of ECM with fibrosis occurs in many tissues as part of the end-organ complications of diabetes (i.e. diabetic nephropathy), and advanced glycosylation end products (AGE) are implicated as one causitive factor in diabetic tissue fibrosis. In addition to being a potent inducer of ECM synthesis and angiogenesis, connective tissue growth factor is increased in tissues from rodent models of diabetes. AGE treatment of primary cultures of CRL-2097 and CRL-1474 nonfetal human dermal fibroblasts resulted in an increase in steady state levels of connective tissue growth factor mRNA as well as protein levels in conditioned medium and cell-associated connective tissue growth factor, while other IGFBP-related proteins were not upregulated by AGE. Thus, AGE upregulates the profibrotic and proangiogenic protein connective tissue growth factor, which may play a role in diabetic complications (Twigg et al., Endocrinology, 2001, 142, 1760-1769).
Diabetic nephropathy is a syndrome occurring in people with diabetes mellitus and characterized by albuminuria, hypertension, and progressive renal insufficiency. Diabetic nephropathy is a common complication in patients with either type 1 or type 2 diabetes mellitus and is recognized to cause severe morbidity and mortality. Structural hallmarks of advanced diabetic nephropathy are glomerulosclerosis and tubulointerstitial fibrosis leading to kidney failure. Current therapies include ACE inhibitors and angiotensin II receptor blockers, both of which are not justified for blanket use among all newly diagnosed patients since only 30-40% will develop progressive renal disease and the long term side effects of these drugs are unknown.
In addition to the need for safe and effective treatments for diabetes is a need for a reliable method to accurately predict, at early stages of disease, which diabetic patients will develop nephropathy and progress to kidney failure. Persistent microalbuminuria is regarded as a predictor of established vascular damage and an indicator of incipient nephropathy. Studies of renal biopsies from patients with type 1 diabetic nephropathy demonstrate an increase in expression of CTGF in renal tissue exhibiting microalbuminuria and nephropathy, relative to normal control tissues (Adler et al., Kidney Int., 2001, 60, 2330-2336), suggesting CTGF is not only a mediator of diabetic nephropathy, but could be used as a marker for the development of disease (Riser et al., Kidney Int., 2003, 64, 451-458).
In a murine lung fibrosis model, an increase in connective tissue growth factor mRNA expression is also induced by bleomycin, a known lung fibrogenic agent (Lasky et al., Am. J. Physiol., 1998, 275, L365-371), as well as in bronchoalveolar lavage cells from patients with idiopathic pulmonary fibrosis and pulmonary sarcoidosis, in comparison to healthy nonsmoking control subjects, indicating that connective tissue growth factor is involved in the fibroproliferative response to injury (Allen et al., Am. J. Respir. Cell Mol. Biol., 1999, 21, 693-700). Similarly, in an experimental model of proliferative glomerulonephritis, connective tissue growth factor mRNA expression was strongly increased in extracapillary and mesangial proliferative lesions and in areas of periglomerular fibrosis. The early glomerular connective tissue growth factor overexpression coincided with a striking upregulation of TGF-β proteins, and the kinetics of connective tissue growth factor expression strongly suggest a role in glomerular repair, possibly downstream of TGF-beta in this model of transient renal injury (Ito et al., J. Am. Soc. Nephrol., 2001, 12, 472-484).
Disclosed and claimed in U.S. Pat. No. 5,876,730 is a substantially pure or isolated polypeptide characterized as having an amino acid sequence corresponding to the carboxy terminal amino acids of a connective tissue growth factor (CTGF) protein, wherein the polypeptide has an amino acid sequence beginning at amino acid residue 247 or 248 from the N-terminus of connective tissue growth factor, an isolated polynucleotide sequence encoding the connective tissue growth factor polypeptide, a recombinant expression vector which contains said polynucleotide, a host cell containing said expression vector, and a pharmaceutical composition comprising a therapeutically effective amount of connective tissue growth factor polypeptide in a pharmaceutically acceptable carrier. Antisense oligonucleotides are generally disclosed (Brigstock and Harding, 1999).
Disclosed and claimed in U.S. Pat. Nos. 5,783,187; 5,585,270; 6,232,064; 6,150,101; 6,069,006 and PCT Publication WO 00/35936 are an isolated polynucleotide encoding the connective tissue growth factor polypeptide, expression vectors, host cells stably transformed or transfected with said vectors; an isolated polynucleotide comprising 5′ untranslated regulatory nucleotide sequences isolated from upstream of connective tissue growth factor, wherein said untranslated regulatory nucleotide sequences comprises a transcriptional and translational initiation region and wherein said sequence is a TGF-beta responsive element; an isolated nucleic acid construct comprising a non-coding regulatory sequence isolated upstream from a connective tissue growth factor (CTGF) gene, wherein said non-coding regulatory sequence is operably associated with a nucleic acid sequence which expresses a protein of interest or antisense RNA, wherein said nucleic acid sequence is heterologous to said non-coding sequence; and a fragment of connective tissue growth factor (CTGF) polypeptide having the ability to induce ECM synthesis, collagen synthesis and/or myofibroblast differentiation, comprising an amino acid sequence encoded by at least exon 2 or exon 3 of said polypeptide. Further claimed is a method for identifying a composition which affects TGF-beta-induced connective tissue growth factor expression, and a method of diagnosing a pathological state in a subject suspected of having a pathology selected from the group consisting of fibrotic disease and atherosclerosis, the method comprising obtaining a sample suspected of containing connective tissue growth factor, whereby detecting a difference in the level of connective tissue growth factor in the sample from the subject as compared to the level of connective tissue growth factor in the normal standard sample is diagnostic of a pathology characterized by a cell proliferative disorder associated with connective tissue growth factor in the subject. Further claimed is a method for ameliorating a cell proliferative disorder associated with connective tissue growth factor, comprising administering to a subject having said disorder, at the site of the disorder, a composition comprising a therapeutically effective amount of an antibody or fragment thereof that binds to connective tissue growth factor, wherein said antibody or fragment thereof does not bind to PDGF. Antisense oligonucleotides are generally disclosed (Grotendorst, 2000; Grotendorst and Bradham, 2001; Grotendorst and Bradham, 2000; Grotendorst and Bradham, 1996; Grotendorst and Bradham, 1998; Grotendorst and Bradham, 2000).
Disclosed and claimed in PCT Publication WO 99/66959 is a device for promoting neuronal regeneration, comprising a gene activated matrix comprising a biocompatible matrix and at least one neuronal therapeutic encoding agent having an operably linked promoter device, wherein the neuronal therapeutic encoding agent encodes an inhibitor of neuronal cell growth, and wherein the inhibitor of neuronal cell growth is selected from the group consisting of NFB42, TGF-beta, connective tissue growth factor (CTGF), and macrophage migration inhibitory factor (MIF), and wherein the neuronal therapeutic encoding agent is selected from the group consisting of a nucleic acid molecule, a vector, an antisense nucleic acid molecule and a ribozyme (Baird et al., 1999).
Disclosed and claimed in PCT Publication WO 00/27868 is a substantially pure connective tissue growth factor polypeptide or functional fragments thereof, an isolated polynucleotide sequence encoding said polypeptide, said polynucleotide sequence wherein T can also be U, a nucleic acid sequence complementary to said polynucleotide sequence, and fragments of said sequences that are at least 15 bases in length and that will hybridize to DNA which encodes the amino acid sequence of the connective tissue growth factor protein under moderate to highly stringent conditions. Further claimed is an expression vector including said polynucleotide, a host cell stably transformed with said vector, an antibody that binds to said polypeptide, and a method for producing said polypeptide. Further claimed is a method for inhibiting the expression of connective tissue growth factor in a cell comprising contacting the cell with a polynucleotide which binds to a target nucleic acid in the cell, wherein the polynucleotide inhibits the expression of connective tissue growth factor in the cell, wherein the polynucleotide is an antisense polynucleotide, as well as a kit for the detection of connective tissue growth factor expression comprising a carrier means being compartmentalized to receive one or more containers, comprising at least one container containing at least one antisense oligonucleotide that binds to connective tissue growth factor (Schmidt et al., 2000).
Disclosed and claimed in PCT Publication WO 00/13706 is a method for treating or preventing fibrosis, the method comprising administering to a subject in need an effective amount of an agent that modulates, regulates or inhibits the expression or activity of connective tissue growth factor or fragments thereof, and wherein the agent is an antibody, an antisense oligonucleotide, or a small molecule. The method is directed to treating kidney fibrosis and associated renal disorders, in particular, complications associated with diabetes and hypertension (Riser and Denichili, 2000).
Disclosed and claimed in PCT Publication WO 01/29217 is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from a group comprising NOV1, NOV2 (connective tissue growth factor), and NOV3, a mature form or variant of an amino acid sequence selected from said group, as well as a nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from said group as well as mature and variant forms or fragments of said polypeptides, and the complement of said nucleic acid molecule. Antisense oligonucleotides are generally disclosed (Prayaga et al., 2001).
There are currently no known therapeutic agents that effectively inhibit the synthesis of connective tissue growth factor and to date, but investigative strategies aimed at modulating connective tissue growth factor function have involved the use of sodium butyrate (NaB), function blocking antibodies and antisense oligonucleotides.
NaB is a dietary micronutrient. Dietary factors are believed to play an important role in both the development and prevention of human cancers, including breast carcinoma. NaB is a major end product of digestion of dietary starch and fiber, and is a potent growth inhibitor that initiates cell differentiation of many cell types in vitro. NaB exerts its biological effects, in part, as a histone deacetylase inhibitor in mammary epithelial cells, induces apoptotic cell death in Hs578T estrogen-non-responsive human breast cancer cells, and can activate different genes involved in cell cycle arrest depending on cell type. NaB specifically upregulates the expression of connective tissue growth factor in a dose-dependent manner, stimulating an increase in both mRNA and protein levels in both cancerous and non-cancerous mammary cells (Tsubaki et al., J. Endocrinol., 2001, 169, 97-110).
TGF-beta has the unique ability to stimulate growth of normal fibroblasts in soft agar, a property of transformed cells. Connective tissue growth factor cannot induce this anchorage-independent growth normal rat kidney (NRK) fibroblasts, but connective tissue growth factor synthesis and action are essential for TGF-β-induced anchorage-independence. Antibodies to connective tissue growth factor specifically blocked TGF-beta-induced anchorage-independent growth, and NRK fibroblasts transformed with a construct expressing the connective tissue growth factor gene in the antisense orientation were not responsive to TGF-beta in the anchorage-independent growth assay (Kothapalli et al., Cell Growth. Differ., 1997, 8, 61-68). These CTGF-antisense expressing NRK cells were also used to show that TGF-beta-stimulated collagen synthesis is mediated by connective tissue growth factor, indicating that connective tissue growth factor may be a useful target for antifibrotic therapies (Duncan et al., Faseb J., 1999, 13, 1774-1786).
The 3′-untranslated region (UTR) of the human connective tissue growth factor cDNA bears several consensus sequences for regulatory elements. When the 3′-UTR was fused downstream of a reporter gene, it was found to act as a strong cis-acting repressive element, and the antisense 3′-UTR had a similar, but stronger effect. (Kubota et al., FEBS Lett., 1999, 450, 84-88). Comparison of the human and mouse connective tissue growth factor 3′-UTRs revealed a conserved small segment of 91 bases. This region was amplified by RT-PCR from NIH3T3 mouse fibroblasts and used to make a chimeric fusion construct for analysis of its repressive effects. The mouse connective tissue growth factor 3′-UTR in either the sense or the antisense orientation had a strong repressive effect on transcription of the reporter gene, indicating an orientation independence of this regulatory element (Kondo et al., Biochem. Biophys. Res. Commun., 2000, 278, 119-124).
A phosphorothioate antisense oligonucleotide, 16 nucleotides in length and targeted to the translation initiation start site, was used to inhibit expression of connective tissue growth factor and suppress proliferation and migration of bovine aorta vascular endothelial cells in culture (Shimo et al., J. Biochem. (Tokyo), 1998, 124, 130-140). This antisense oligonucleotide was also used to show that connective tissue growth factor induces apoptosis in MCF-7 human breast cancer cells and that TGF-beta-induced apoptosis is mediated, in part, by connective tissue growth factor (Hishikawa et al., J. Biol. Chem., 1999, 274, 37461-37466). The same antisense oligonucleotide was also found to inhibit the TGF-beta-mediated activation of caspase 3 and thus to inhibit induction of TGF-beta-mediated apoptosis in human aortic smooth muscle cells (HASC) (Hishikawa et al., Eur. J. Pharmacol., 1999, 385, 287-290). This antisense oligonucleotide was also used to block connective tissue growth factor expression and demonstrate that high blood pressure upregulates expression of connective tissue growth factor in mesangial cells, which in turn enhances ECM protein production and induces apoptosis, contributing to the remodeling of mesangium and ultimately glomerulosclerosis (Hishikawa et al., J. Biol. Chem., 2001, 276, 16797-16803).
Consequently, there remains a long felt need for additional agents capable of effectively inhibiting connective tissue growth factor function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of connective tissue growth factor expression.
The present invention provides compositions and methods for modulating connective tissue growth factor expression in cells and in vivo.